Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press A novel form of RNA double helix based on G·U and C·A+ wobble base pairing ANKUR GARG1,2 and UDO HEINEMANN1,2 1Crystallography, Max-Delbrück Center for Molecular Medicine in the Helmholtz Association, 13125 Berlin, Germany 2Institute for Chemistry and Biochemistry, Freie University Berlin, 14195 Berlin, Germany ABSTRACT Wobble base pairs are critical in various physiological functions and have been linked to local structural perturbations in double- helical structures of nucleic acids. We report a 1.38-Å resolution crystal structure of an antiparallel octadecamer RNA double helix in overall A conformation, which includes a unique, central stretch of six consecutive wobble base pairs (W helix) with two G·U and four rare C·A+ wobble pairs. Four adenines within the W helix are N1-protonated and wobble-base-paired with the opposing cytosine through two regular hydrogen bonds. Combined with the two G·U pairs, the C·A+ base pairs facilitate formation of a half turn of W-helical RNA flanked by six regular Watson–Crick base pairs in standard A conformation on either side. RNA melting experiments monitored by differential scanning calorimetry, UV and circular dichroism spectroscopy demonstrate that the RNA octadecamer undergoes a pH-induced structural transition which is consistent with the presence of a duplex with C·A+ base pairs at acidic pH. Our crystal structure provides a first glimpse of an RNA double helix based entirely on wobble base pairs with possible applications in RNA or DNA nanotechnology and pH biosensors. Keywords: RNA double helix; wobble base pairing; wobble helix; adenine N1 protonation; pH-dependent structural variation INTRODUCTION angles to form two stable hydrogen bonds (Hunter et al. 1987; Puglisi et al. 1990). The formation of two isosteric hy- Base-pairing in most nucleic acids follows the Watson–Crick drogen bonds in a C·A wobble pair, however, is not possible pairing rules. Non-Watson–Crick base-pairing, however, is with bases in their standard configuration. Here, the N1(A)– also prevalent and plays crucial roles for various physiological O2(C) hydrogen bond can only form if either the proton- functions (Deng and Sundaralingam 2000; Masquida and ation or the tautomeric state of the participating bases are Westhof 2000; Varani and McClain 2000). Among all non- changed. A first model proposes hydrogen-bond stabilization Watson–Crick pairs, the G·U wobble is the most studied by rare amino–imino tautomers of cytosine or adenine pair that has been shown to be highly conserved in the accep- (Saenger 1983; Hunter et al. 1986, 1987; Russo et al. 1998; tor helix of tRNAAla, common in other tRNAs (Sprinzl et al. Masoodi et al. 2016). Similarly, a second model suggests pro- 1998) and rRNA (Gautheret et al. 1995), and critical tonation of cytosine N3 simultaneously with a tautomeric for RNA–protein recognition (Hou and Schimmel 1988; shift in the adenine base (Supplemental Fig. S1). Cytosine McClain and Foss 1988) and splice-site selection in group I N3 protonation has been invoked in U6 RNA loop structure introns (Cech 1987; Strobel and Cech 1995). Among other formation in Trypanosoma brucei and Crithidia fasciculata mismatches, C·A+ wobble pairs are less frequently observed; (Huppler et al. 2002) and the DNA–triostin-A interaction however, they have been shown to be isosteric with and capa- (Quigley et al. 1986). However, whereas the transient forma- ble of substituting G·U pairs in some cases (Samuelsson et al. tion of base pairs involving the rare enol tautomers of 1983; Doudna et al. 1989; Gautheret et al. 1995; Masquida guanine and thymine was recently demonstrated by NMR and Westhof 2000). methods (Kimsey et al. 2015), experimental proof of C·A G·U and C·A+ pairs are significantly different from Watson– pairs with adenine imino tautomers is lacking, to the best Crick base pairs, and they present opportunities for specific of our knowledge. recognition either by generating local irregularity of helical The most frequently invoked model for C·A mismatch structure or by introducing electrostatic variations in the formation proposes protonation of adenine N1, which would grooves. G·U wobble base pairs are thermodynamically fa- vorable and require only slight adjustment in base pair λ © 2018 Garg and Heinemann This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see Corresponding author: [email protected] http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is avail- Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.064048. able under a Creative Commons License (Attribution-NonCommercial 4.0 117. International), as described at http://creativecommons.org/licenses/by-nc/4.0/. RNA 24:209–218; Published by Cold Spring Harbor Laboratory Press for the RNA Society 209 Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Garg and Heinemann allow a second hydrogen bond to form with cytosine O2 as tioned a partial RNA model into the unit cell with log- acceptor atom (Hunter et al. 1986, 1987). In aqueous solu- likelihood gain (LLG) and translation function Z-score tion, adenosine exists in three different mono-protonation (TFZ) of 54.4 and 5.3, respectively. One round of automated states with pKa values of 3.64, −1.53, and −4.02 for N1, model building and refinement was performed using N7, and N3 protonation (Saenger 1983; Kapinos et al. Autobuild wizard, which, after fitting the correct oligonucle- 2011), and the protonation propensity of adenosine N1 was otide sequence and refinement, yielded Rwork/Rfree of 23.7%/ reported as being 96.1% over N7 and N3 protonation states 24.0%. Bond length and angle restraints combined with base- (Kapinos et al. 2011). Also, adenine N1 protonation has planarity and hydrogen-bonding restraints for base pairs been observed in poly(rA) fibers (Rich et al. 1961), oligo were used simultaneously with anisotropic displacement (rA) stretches in crystal structures (Gleghorn et al. 2016), factor restraints over the whole refinement process, which and an NMR structure of poly(dA) (Chakraborty et al. yielded a final Rwork/Rfree of 12.9%/15.1% (Table 1). 2009), allowing the oligo(A) to form either a parallel-strand- The RNA molecule crystallized in space group P6322, ed double helix (π-helix) at acidic pH or a single-stranded forming an 18-bp antiparallel RNA double helix with two helical structure at neutral pH. identical strands related by crystallographic symmetry. In addition, the presence of adenine N1 protonation in These duplexes are stacked in head-to-tail fashion to generate + isolated or tandem C·A wobble base pairs is confirmed by a pseudo-continuous RNA column around the 63 screw axis several crystal (Hunter et al. 1986, 1987; Jang et al. 1998; along the crystallographic c-axis (Supplemental Fig. S2A). Pan et al. 1998) and NMR (Puglisi et al. 1990; Durant and The helical stacks are stabilized by eight well-coordinated Davis 1999; Huppler et al. 2002) structures of different oligo- water molecules present in the minor groove at the helical nucleotides. Interestingly, tandem C·A+/A+·C pairs exhibit junction. The eight waters are symmetrically arranged and cross-strand purine stacking due to the near 0° helical twist, form a hydrogen-bonding network that stabilizes the helical which is compensated by a twist increase by 10°–15° at the arrangement by forming specific hydrogen bonds with all adjacent Watson–Crick pairs (Jang et al. 1998), while an iso- four sugar 2′OH, N3, and O2 atoms of adenine and uridine, lated C·A+ pair causes little helical irregularity, suggesting respectively (Supplemental Fig. S2B). that neighboring base pairs are efficient in maintaining over- all helical geometry when more than one C·A+ pair is present. Geometry of RNA double helix Although several structures for base pair mismatches are available showing G·U or C·A+ wobble pairs in isolation, in In the crystal, two RNA strands form an antiparallel double tandem or with other mismatches, both wobble pairs have helical structure with 18 bp (Fig. 1). The helical parameters never been shown together in a crystal structure. Only one for the full-length RNA structure (fl helix) suggest that the NMR structure of an RNA hairpin with both G·U and double helix exhibits A-RNA conformation with helical C·A+ pairs is available that shows these wobble pairs in isola- periodicity of 10.96 bp, similar to canonical A-RNA, which tion (Puglisi et al. 1990). has 11 bp per helical turn (Table 2; Schindelin et al. 1995; Here we present a high-resolution crystal structure of an Olson et al. 2001). The RNA double helix can be divided 18-bp antiparallel RNA double helix, which includes one into three helical segments of 6 bp each with the first and half helical turn of W helix, comprising six contiguous wob- third segments (WC helix) having a total of 12 bases paired ble base pairs at the center flanked by half turns of standard A-form RNA on either side. The W-helical stretch is formed by four C·A+ and two G·U wobble base pairs. The geometry TABLE 1. X-ray data collection and refinement statistics of the novel form of RNA helix is described in detail with a Data collection statistics focus on the central wobble pairs. In addition, pH-dependent Space group P6322 variations in the RNA structure are probed by various a, b, c (Å) 45.00, 45.00, 94.20 Resolution (Å) 38.97–1.38 (1.46–1.38) biophysical methods.